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Bladder cancer (BC) is the sixth most commonly diagnosed tumor worldwide. DNA repair capacity (DRC) refers to the ability of a cell to protect the integrity of the genome and DNA repair pathways have been implicated in BC risk. It has been observed that individuals with low DRC tend to accumulate more damage than those with a more efficient DRC. This inter-individual variability is modulated by the genetic background, as well as differential gene expression and epigenetic regulation. We aimed at studying the relationship between DRC and DNA damage (evaluated by H2AX phosphorylation and micronucleus assays) and BC risk and clinical outcome, integrating with gene expression and epigenetic profile data in 159 BC cases and 159 matched controls, enrolled in the Turin Bladder Cancer Study (TBCS). We investigated ã-H2AX phosphorylation levels and MN frequencies in cryopreserved peripheral blood mononuclear cells. We found significant differences in micronuclei and nuclear buds frequencies, with higher number of these damages in cases compared to controls (p = 0.0002 and p = 0.002 respectively). On the other hand, we observed a significant association between ã-H2AX basal levels and risk of disease recurrence/progression in both BC patients as a whole and the subgroup of non-muscle invasive BC (NMIBC) (for all BC HR 0.70, 95% CI 0.52-0.94, p = 0.02; for NMIBC HR 0.68, 95% CI 0.50-0.92, p = 0.01): this suggests a protective effect of DNA double strand breaks signalling in terms of preventing BC recurrence or progression. In order to evaluate the genetic and epigenetic role in modulation of DRC we performed whole genome methylation and gene expression analyses on the same BC cases and controls. Preliminary analyses on methylation levels did not show any significant difference between cases and controls. Two metalloproteinases (MMP23A and MMP23B) resulted significantly under-expressed in BC compared to healthy controls (logFC = -0.23, p = 0.01; logFC = -0.37, p = 0.007, respectively). Interestingly, the expression levels of these genes were also significantly correlated with the relative CpGs methylation. Further analyses focusing on the integration of whole genome data with DRC assays are ongoing to unravel new prognostic biomarkers of disease. Citation Format: Giuseppe Matullo, Clara Viberti, Barbara Pardini, Alessandra Allione, Simonetta Guarrera, Valentina Turinetto, Claudia Giachino, Giovanni Fiorito, Alessio Naccarati, Alessia Russo, Rossana Critelli, Paolo Destefanis, Marco Oderda, Paolo Gontero, Paolo Vineis, Carlotta Sacerdote. DNA repair capacity, chromosomal damage, methylation and gene expression levels in bladder cancer: An integrated analysis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 778.

Bladder cancer (BC), which is the fourth most common malignancy among men in the Western world, has a typical aetiology characterized by a multistep carcinogenesis, reflecting that multiple lesions in the DNA are required for tumour development. DNA repair capacity (DRC) is a complex marker comprising the sum of several factors such as gene variants, gene expression, stability of gene products, and effect of inhibitors/stimulators and might account for different susceptibility of developing this cancer. Individuals with low DRC will tend to accumulate more damage than those who have a better ability to repair such damage. The identification of patients with particular DRC and at high-risk of BC and/or recurrence of the disease could help in personalizing both surveillance and treatment. We aimed at studying the relationship between DRC evaluated by H2AX phosphorylation assays and BC, integrating gene expression and epigenetic profile data (methylation levels and microRNA expression) in cryopreserved lymphocytes from 159 BC cases and 159 controls matched by age and smoking habits. We observed a significant association between γ-H2AX basal levels and H2AX dephosphorylation capacity and risk of BC recurrence. Patients with high basal DSB signaling had a better event-free survival (HR 0.35, 95% CI 0. 19-0.63, p = 0.0005). Additionally, there was an association when considering the percentage of dephosphorylation after 3h divided into two categories (above and under the calculated best cut-off). Interestingly, when compared with the lower category (“slow repair”), the other group (“fast repair”) had a better event-free survival (HR 0.41, 95%CI 0.23-0.72, p = 0.002). Our data suggest that γ-H2AX can be considered as a possible molecular biomarker to identify patients with a higher risk of BC recurrence. A subgroup of cases and controls (39 and 44, respectively) was also analyzed for evaluating their gene expression (Illumina HumanHT-12 Expression BeadChip array) and methylation profile data (Illumina HumanMethylation450 BeadChip array). We found a set of CpG islands that were differentially methylated among cases and controls and a set of genes whose expression was deregulated among different subgroups (e.g. cases vs healthy controls, controls vs BC patients with muscle invasive tumor, etc). We performed an integrated analysis on the genotype/phenotype correlation in the analysed BC cases and healthy controls provided with detailed description of the follow-up for response to therapy/recurrence/survival. This integrated approach will help in elucidating the role of DRC (and its determinants) as predictive and prognostic marker and to identify DNA repair phenotypic assays to be performed in blood cells as non-invasive predictive method. Results will be presented at the AACR Annual Meeting. Citation Format: Barbara Pardini, Alessandra Allione, Simonetta Guarrera, Valentina Turinetto, Giovanni Fiorito, Clara Viberti, Alessia Russo, Paolo Vineis, Carlotta Sacerdote, Claudia Giachino, Giuseppe Matullo. H2AX phosphorylation assays, gene expression and epigenomic profiles as markers in bladder cancer: an integrated approach. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4615. doi:10.1158/1538-7445.AM2015-4615

In Europe, bladder cancer (BC) is the sixth most commonly diagnosed tumor and the second most common cause of death among patients with genitourinary tract malignancies. The ability to repair DNA damage is strongly associated with the risk of cancer and inter-individual variation in DNA repair capacity (DRC) might account for different susceptibility of developing cancer. DRC represents a complex marker comprising the sum of several factors such as gene variants, gene expression, stability of gene products, and effect of inhibitors/stimulators. Individuals with low DRC will tend to accumulate more damage than those who have a better ability to repair such damage. This variability is modulated by the genetic background to which SNPs/haplotype combinations in DNA repair genes, as well as epigenetic regulation are likely to contribute. Phenotypic assays to evaluate DNA repair activity (in particular NER comet assay, H2AX phosphorylation and micronucleus assay) were performed on cryopreserved lymphocytes from 159 cases (collected before treatment) and 159 controls matched by age and smoking habits, enrolled in Turin Bladder Cancer Study (TBCS). We aimed at studying the relationship between DRC (evaluated by comet assay, micronuclei and H2AX phosphorylation assay) and bladder cancer integrating, gene expression and epigenetic profile data (methylation levels and microRNA expression). In particular, we performed an integrated analysis on the genotype/phenotype correlation in a population of bladder cancer cases and controls provided with detailed description of the follow-up for response to therapy/recurrence/survival. This integrated approach will help in elucidating the role of DRC (and its determinants) as predictive and prognostic marker and to identify combined biomarkers to be measured in blood cells as non-invasive predictive method. Citation Format: Barbara Pardini, Alessandra Allione, Simonetta Guarrera, Valentina Turinetto, Giovanni Fiorito, Fabio Rosa, Alessia Russo, Federica Modica, Claudia Giachino, Paolo Vineis, Carlotta Sacerdote, Giuseppe Matullo. DNA repair capacity, gene expression and epigenomic profiles in bladder cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5367. doi:10.1158/1538-7445.AM2014-5367

Individual DNA repair capacity is modulated by the genetic background to which hundreds of single nucleotide polymorphisms (SNPs) or haplotype combinations in DNA repair genes are likely to contribute. A large number of studies have been published on the association between DNA repair gene polymorphisms and cancer risk, whereas only few small studies investigated the relation between DNA repair phenotypic assays and cancer risk or DNA repair polymorphisms. In order to identify functional SNPs/haplotypes modulating DNA repair phenotypes and to investigate their potential impact on cancer risk, we have performed a genotype-phenotype correlation study evaluating levels of DNA damage and DNA repair capacity in blood cells treated at low doses of different DNA damaging agents. We investigated the association between genotypes/haplotypes and the DNA repair capacity measured with the following DNA repair phenotypic assays on cryopreserved lymphocytes from 225 healthy individuals: Comet assay after treatment with BPDE, phosphorylation of histone H2AX after treatment with γ-rays, OGG1 glycosylase activity, OGG1 and ERCC1 expression levels. Sixty-five DNA repair genes and a total of 768 SNPs have been selected and analysed on the 225 DNA samples by using a custom GoldenGate SNP chip (Illumina). Genotype-phenotype correlation analysis identified some new possible functional variants and haplotypes. We also tested gene-gene interactions, comparing results with existing prior knowledge from different publicly available databases and crossing functional evidence with information on gene expression levels and protein-protein interaction. A confirmation of some already described interplays between different DNA repair pathways have been obtained from SNP analysis and a suggestion of new possible cross-talks have been identified that need further experimental validation. Our results can contribute to identify subjects carrying combinations of less efficient DNA repair variants/haplotypes possibly at increased cancer risk or who can better benefit from anti-cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2739. doi:10.1158/1538-7445.AM2011-2739